Accumulating evidence has provided a strong link between progastrin-derived peptides and colorectal cancer. Studies from our group employing gastrin over-expressing and gastrin-deficient mice have demonstrated that progastrin or incompletely processed forms of gastrin can modulate colonic proliferation and intestinal tumorigenesis in the mouse. In addition, published work from our laboratory has shown that the gastrin gene is a direct downstream target of the Wnt (Apc/beta-catenin) signaling pathway. More recently, we have discovered that other oncogenic/tumor suppressor pathways, such as TGF- beta/Smad and Ras can modulate Wnt-dependent regulation of the gastrin promoter. Both TGF- beta and Ras signaling can strongly synergize with Wnt-dependent transactivation of gastrin. While issues of gastrin processing have created obstacles in the past to in vivo studies of gastrin gene expression, we have recently generated gastrin-GFP knock-in mice that show tissue specific expression of GFP. Furthermore, using tagged peptides we have demonstrated highly specific binding of progastrin and G-gly to cell membranes that appears to be distinct from the classic CCK-2 receptor but closely linked to Src/Erk/Stat signaling in colonic epithelial cells. Thus, we hypothesize a role for progastrin as a key link between genetic alterations and activated signaling/proliferation. In order to identify pathways both upstream and downstream of gastrin gene expression in colon cancer, we propose the following three specific aims: (1). Investigate the regulation of the gastrin promoter by oncogenic/tumor suppressor pathways. The roles of TGF- beta and Ras in modulating the action of Wnt signaling on the gastrin promoter will be defined (2). Study the in vivo activation of gastrin using a transgenic (mGAS-EGFP knockin mouse) reporter gene mouse. The mGAS-EGFP-KI mouse will be crossed to a number of cancer models and the role of progastrin in stem cell activation explored. (3). Identify receptors and downstream targets that mediate the effects of progastrin. Labeled progastrin-derived peptides will be used to screen cDNA libraries in order to identify specific receptors mediating progastrin's effects. Overall, these studies will attempt to define the role of progastrin-derived peptides in stem cell regulation and in bridging genetic pathways in colorectal cancer.